Abstract

Introduction

CDK8 is an oncogenic cyclin-dependent kinase that exists as part of the kinase module within the Mediator complex. This complex interacts with the transcription machinery to regulate transcription; signal transduction pathways, including the WNT pathway; and biological processes, such as cell cycle progression. Recently, we identified a series of 3,4,5-trisubstituted pyridines as inhibitors of CDK8 and, its paralogue, CDK19 in colorectal cancer (CRC). Until now, there have been few validated substrates of CDK8/19. Here, we describe a motif-based phospho-proteomic approach, utilizing our 3,4,5-trisubstituted pyridine inhibitors that we have used to identify substrates of CDK8/19. These substrates could represent useful biomarkers for future drug discovery research.

Experimental Outline

We used a COLO205 cell line (COLO205 C4) carrying a TCF/LEF reporter construct responsive to CDK8/19 inhibition and CCT251545, a compound we have previously shown to be a potent and selective inhibitor of CDK8/19. Cells were treated with 350 nM CCT251545 (10 x EC50) for 2 or 6 hours. Proteins were then extracted from treated and control cells, trypsin-digested and immunoprecipitated for phospho-peptide enrichment using proline-directed motifs: PXS*P, S*PXR/K, PXS*PXR/K + T*PE + ST*P + K/HS*P. Potential substrates were identified by LC-MS/MS and validated by immunoprecipitation and western blotting. Substrates were validated in another CRC cell line, SW620, which harbors CRISPR knockouts for CDK8/19.

Summary of Results

LC-MS/MS analyses of COLO205 C4 cell extracts revealed a number of potential CDK8/19 substrates, including some Mediator complex subunits such as MED13, transcriptional coactivators such as HCFC1, and transcription factors such as STAT1. Phosphorylation of STAT1SER727 was the top ranked hit and follow-up studies, in COLO205 C4 cells and xenografts, confirmed repression of STAT1SER727 phosphorylation in the presence of CCT251545. An inactive analogue, CCT251099, and other kinase inhibitors (flavopiridol, KN-93, PD 0325901 and SB 202190) did not block STAT1SER727 phosphorylation. Repression of STAT1SER727 phosphorylation upon treatment with CCT251545 was also observed in SW620 and LS174T CRC cells and xenografts.

Conclusion

A motif-driven, mass spectroscopy-based phospho-proteomic study identified candidate substrates of CDK8/19. Phosphorylation of STAT1SER727 was validated as a useful marker of target engagement in CRC cell lines both in vitro and in vivo.